**2.3. VADS constructed with liposomes formed by self-assembly of phospholipids**

diphtheria toxoid were observed after injection of the liposome vaccines into mice [40, 44, 45]. It is generally accepted that liposomes as a VADS function with the adjuvanticity regardless of the carrying mode of Ags, including entrapment within vesicles, attachment on surfaces, or simply mixing together [46, 47], which allows diverse modifications to be carried out on liposomes without concerning the Ag damage through measures, including PEGylation, decoration with PAMP molecules or the pattern recognition receptor agonists (PRRas), such as lipid A for TLR4, CpG-ODN for TLR9, and synthetic mannose derivatives for C-type receptors on

Vaccine Adjuvant Delivery Systems Constructed Using Biocompatible Nanoparticles Formed…

Notably, multifunctional liposomes have also been developed in combination with novel administration devices to form a VADS which can be employed to enhance immunization efficacy via convenient administration [47]. In particular, Wang's group developed the multifunctional liposome-based VADSs through fabrication of liposomes adorned with TLR4a lipid A and loaded with Ags into biodegradable microneedle arrays, which can efficiently exert penetration of mucosa enhancing topical delivery efficiency [37, 48, 50]. Going further, Wang and coworkers engineered two types of multifunctional liposomes, the 200 nm-sized mannosylated lipid A-liposomes (MLLs) and the 50 nm stealth lipid A-liposomes (SLLs), both

HCO3

species) stimulation, which were caused, respectively, by expansion of CO2

complex procedure for products and the instable entrapment of volatile NH4

forming the proSLL/MLL-constituted microneedle array (proSMMA), which proved able to rapidly recover the initial MLLs and SLLs upon rehydration by tissue fluids [47]. Mice vaccinated with proSMMAs by vaginal mucosa patching established robust Ag-specific humoral and cellular immunity at both systemic and mucosal systems, especially, in the reproductive and intestinal ducts, owing to the action processes involving the facts that the MLLs reconstituted from the administered proSMMAs were mostly taken up by vaccination site-resident DCs for mucosal responses, whereas the smaller SLLs traveled to the dLDs wherein picked up by macrophages for efficient use of Ags. Furthermore, the delivered Ags were displayed by APCs via cross-presented with MHC-I thanks to lysosome escape and ROS (reactive oxygen

both sourcing from the liposome-released NH4

mixed Th1/Th2 type response promoted further by liposomal lipid A and activation of TLR4. Thus, though the large-scale production of the proSMMAs seems still a problem owing to the

the multifunctional VADS constructed with liposomal microneedles for vaginal immunization provides an alternative strategy to elicit immunity against various pathogens, especially, the sexually transmitted ones. Moon et al. fabricated a novel VADS based on a special type of liposomes, which were called interbilayer-crosslinked multilamellar vesicles (ICMVs) and formed by crosslinking headgroups of adjacent phospholipid bilayers within multilamellar vesicles [51]. Further investigation showed that the stable Ag/adjuvant-carried ICMVs rapidly released the loaded cargos in response to catalysis by endolysosomal lipases, and when given to mice elicited robust endogenous T-cell and antibody responses, suggesting ICMVs a stimulus-sensitive VADS which may open up new possibilities for vaccination against infec-

Summarily, liposomes are the most diverse carrier for delivering various agents and can be employed through diverse modifications with various functional molecules to constitute

and then packed together into microneedles,

http://dx.doi.org/10.5772/intechopen.79905

17

gas and induc-

, leading to a

in vesicles,

HCO3

HCO3

APCs [8, 9, 37, 48, 49].

tion of excessive NH4

tious diseases and cancer.

of which were loaded with Ags and NH4

+ /NH3

Liposomes are the phospholipid bilayer-enclosed vesicles and have attracted many research interests in the development of drug delivery system (DDS) as well as VADS ever since its discovery by Bangham et al. in the early 1960s [38]. Liposomes usually consist of one or more concentric lipid bilayers alternating with aqueous spaces [39, 40], with the components of one, or more type of amphiphilic phospholipids such as phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), and sphingomyelin (SM), which though form the frame structure of liposomes and are often supplemented with ingredients, such as cholesterol (CHO) and other charged lipids such as stearylamine (SA), N[1-(2,3-dioleyloxy) propyl]-N,N,Ntriethylammonium (DOTMA), 1,2-dioleyloxy-3-(trimethylammonium propane) (DOTAP), and 3 (N,N,-dimethylaminoethane)-carbamyl cholesterol (DMACHO), which are purposely used for tailoring the property of liposomes. Depending on ambient temperature and the nature of the lipids, the liposome bilayers may exist in either a "fluid" state when the ambient temperature is above the Tc (a gel to liquid crystalline transition temperature―the temperature at which the acyl chains melt) of liposomes, or a "rigid" state when the ambient temperature falls below the Tc of liposomes [40]. However, when CHO is homogenously incorporated into phospholipid bilayers, for example, at the mole ratio of CHO/PC between 1/3 and 2/3, the membrane rigidity may be significantly strengthened, and as a consequence, liposomes are blurred of Tc lowering content leakage. Bearing a common weakness of instability associated with a colloidal system, liposomes are often superficially PEGylated (modification with polyethylene glycol, PEG) to engender a steric stabilization effect, and/or are charged with an appropriate zeta potential value by the incorporated ionic lipids to generate an electrostatic repulsion for preventing aggregation, or for flocculating particles according to DLVO theory. Also, lyophilization is often employed to engender liposomes into a dry entity, which has a high stability completely satisfying the shelf-life requirements for clinical application, as it can be rehydrated to reconstitute the initial vesicles with little cargo leakage just in the presence of disaccharide as an effective lyoprotectant [41].

Liposomes possess numerous distinct properties enabling them to fulfill the functions of an excellent VADS, which can be summarized as aspects including good biocompatibility, high loading capacity for various ingredients, and the ease for preparation and surface decoration to engender specific functions such as targeting delivery, lysosome escape and controlled release [42]. With ability to entrap hydrophilic, lipophilic as well as amphiphilic molecules in the inner aqueous phase or lipid bilayers, liposomes have been formulated for delivering a large range of therapeutic agents, including small molecules, DNA/RNA fragments, peptides, and even proteins with a large molecular weight (MW), which exhibit respective therapeutic activities [39]. Also, liposomes are frequently employed as a VADS fitting diverse immunization routes, including intravenous, intramuscular, subcutaneous, intranasal, oral uptake, pulmonary inhalation, and topical skin or mucosal administration, for delivering vaccines to resolve problems associated with free Ags, for instances, averting premature inactivation caused by environmental chemicals, and ensuring Ags to approach APCs and even the intracellular organelles without off-targets [43]. In particular, beneficial for acting as a VADS, liposomes possess the intrinsic adjuvant properties as established by Gregoriadis and coworkers in as early as 1974 when strong humoral immune responses to liposome-entrapped diphtheria toxoid were observed after injection of the liposome vaccines into mice [40, 44, 45]. It is generally accepted that liposomes as a VADS function with the adjuvanticity regardless of the carrying mode of Ags, including entrapment within vesicles, attachment on surfaces, or simply mixing together [46, 47], which allows diverse modifications to be carried out on liposomes without concerning the Ag damage through measures, including PEGylation, decoration with PAMP molecules or the pattern recognition receptor agonists (PRRas), such as lipid A for TLR4, CpG-ODN for TLR9, and synthetic mannose derivatives for C-type receptors on APCs [8, 9, 37, 48, 49].

**2.3. VADS constructed with liposomes formed by self-assembly of phospholipids**

16 Immunization - Vaccine Adjuvant Delivery System and Strategies

ence of disaccharide as an effective lyoprotectant [41].

Liposomes are the phospholipid bilayer-enclosed vesicles and have attracted many research interests in the development of drug delivery system (DDS) as well as VADS ever since its discovery by Bangham et al. in the early 1960s [38]. Liposomes usually consist of one or more concentric lipid bilayers alternating with aqueous spaces [39, 40], with the components of one, or more type of amphiphilic phospholipids such as phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), and sphingomyelin (SM), which though form the frame structure of liposomes and are often supplemented with ingredients, such as cholesterol (CHO) and other charged lipids such as stearylamine (SA), N[1-(2,3-dioleyloxy) propyl]-N,N,Ntriethylammonium (DOTMA), 1,2-dioleyloxy-3-(trimethylammonium propane) (DOTAP), and 3 (N,N,-dimethylaminoethane)-carbamyl cholesterol (DMACHO), which are purposely used for tailoring the property of liposomes. Depending on ambient temperature and the nature of the lipids, the liposome bilayers may exist in either a "fluid" state when the ambient temperature is above the Tc (a gel to liquid crystalline transition temperature―the temperature at which the acyl chains melt) of liposomes, or a "rigid" state when the ambient temperature falls below the Tc of liposomes [40]. However, when CHO is homogenously incorporated into phospholipid bilayers, for example, at the mole ratio of CHO/PC between 1/3 and 2/3, the membrane rigidity may be significantly strengthened, and as a consequence, liposomes are blurred of Tc lowering content leakage. Bearing a common weakness of instability associated with a colloidal system, liposomes are often superficially PEGylated (modification with polyethylene glycol, PEG) to engender a steric stabilization effect, and/or are charged with an appropriate zeta potential value by the incorporated ionic lipids to generate an electrostatic repulsion for preventing aggregation, or for flocculating particles according to DLVO theory. Also, lyophilization is often employed to engender liposomes into a dry entity, which has a high stability completely satisfying the shelf-life requirements for clinical application, as it can be rehydrated to reconstitute the initial vesicles with little cargo leakage just in the pres-

Liposomes possess numerous distinct properties enabling them to fulfill the functions of an excellent VADS, which can be summarized as aspects including good biocompatibility, high loading capacity for various ingredients, and the ease for preparation and surface decoration to engender specific functions such as targeting delivery, lysosome escape and controlled release [42]. With ability to entrap hydrophilic, lipophilic as well as amphiphilic molecules in the inner aqueous phase or lipid bilayers, liposomes have been formulated for delivering a large range of therapeutic agents, including small molecules, DNA/RNA fragments, peptides, and even proteins with a large molecular weight (MW), which exhibit respective therapeutic activities [39]. Also, liposomes are frequently employed as a VADS fitting diverse immunization routes, including intravenous, intramuscular, subcutaneous, intranasal, oral uptake, pulmonary inhalation, and topical skin or mucosal administration, for delivering vaccines to resolve problems associated with free Ags, for instances, averting premature inactivation caused by environmental chemicals, and ensuring Ags to approach APCs and even the intracellular organelles without off-targets [43]. In particular, beneficial for acting as a VADS, liposomes possess the intrinsic adjuvant properties as established by Gregoriadis and coworkers in as early as 1974 when strong humoral immune responses to liposome-entrapped Notably, multifunctional liposomes have also been developed in combination with novel administration devices to form a VADS which can be employed to enhance immunization efficacy via convenient administration [47]. In particular, Wang's group developed the multifunctional liposome-based VADSs through fabrication of liposomes adorned with TLR4a lipid A and loaded with Ags into biodegradable microneedle arrays, which can efficiently exert penetration of mucosa enhancing topical delivery efficiency [37, 48, 50]. Going further, Wang and coworkers engineered two types of multifunctional liposomes, the 200 nm-sized mannosylated lipid A-liposomes (MLLs) and the 50 nm stealth lipid A-liposomes (SLLs), both of which were loaded with Ags and NH4 HCO3 and then packed together into microneedles, forming the proSLL/MLL-constituted microneedle array (proSMMA), which proved able to rapidly recover the initial MLLs and SLLs upon rehydration by tissue fluids [47]. Mice vaccinated with proSMMAs by vaginal mucosa patching established robust Ag-specific humoral and cellular immunity at both systemic and mucosal systems, especially, in the reproductive and intestinal ducts, owing to the action processes involving the facts that the MLLs reconstituted from the administered proSMMAs were mostly taken up by vaccination site-resident DCs for mucosal responses, whereas the smaller SLLs traveled to the dLDs wherein picked up by macrophages for efficient use of Ags. Furthermore, the delivered Ags were displayed by APCs via cross-presented with MHC-I thanks to lysosome escape and ROS (reactive oxygen species) stimulation, which were caused, respectively, by expansion of CO2 gas and induction of excessive NH4 + /NH3 both sourcing from the liposome-released NH4 HCO3 , leading to a mixed Th1/Th2 type response promoted further by liposomal lipid A and activation of TLR4. Thus, though the large-scale production of the proSMMAs seems still a problem owing to the complex procedure for products and the instable entrapment of volatile NH4 HCO3 in vesicles, the multifunctional VADS constructed with liposomal microneedles for vaginal immunization provides an alternative strategy to elicit immunity against various pathogens, especially, the sexually transmitted ones. Moon et al. fabricated a novel VADS based on a special type of liposomes, which were called interbilayer-crosslinked multilamellar vesicles (ICMVs) and formed by crosslinking headgroups of adjacent phospholipid bilayers within multilamellar vesicles [51]. Further investigation showed that the stable Ag/adjuvant-carried ICMVs rapidly released the loaded cargos in response to catalysis by endolysosomal lipases, and when given to mice elicited robust endogenous T-cell and antibody responses, suggesting ICMVs a stimulus-sensitive VADS which may open up new possibilities for vaccination against infectious diseases and cancer.

Summarily, liposomes are the most diverse carrier for delivering various agents and can be employed through diverse modifications with various functional molecules to constitute different types of multifunctional VADS satisfying different vaccination requirements. As a proved by numerous experiments, at least in animal models, these multifunctional liposome VADSs are highly effective in both targeting delivery of vaccine to APCs and enhancing Ag presentation by APCs to related T-cells to set up the Ag-specific immunity against pathogens, fulfilling a dual function of adjuvancy and delivery for vaccines [7, 37, 47–49].

differentiated and proliferated from relevant precursors [61] through MyD88 (the myeloid differentiation primary response gene 88) adapter protein-expression pathway, as revealed by Wilson et al. [62]. Notably, ISCOMs were upgraded by Schiött and coworkers to the next generation VADS, denoted Posintro™, which were cationic NPs formulated with cholesterol, DC-cholesterol (3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl) cholesterol hydrochloride), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), Quil A and HBsAg in the weight ratio of 3:1:4:20:5, engendering a new HBV vaccine of Posintro™-HBsAg [63]. In the intradermally (i.d.) immunized animal models of mice and guinea pigs, Posintro™-HBsAg induced the strong response with high titers of HBsAg-specific antibody and high levels of cytotoxic T lymphocyte (CTL), demonstrating that Posintro™-HBsAg is promising both for the protec-

Vaccine Adjuvant Delivery Systems Constructed Using Biocompatible Nanoparticles Formed…

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19

Notably, to develop effective vaccines against the deadly Ebola virus (EBV), which causes a fatal hemorrhagic fever in humans with a mortality of around 50%, as evidenced by the 2014–2016 West Africa Ebola epidemic which claimed 11,310 lives in 28,600 infection cases [64], Bengtsson et al. engineered the 2014 EBV Makona strain glycoprotein (EBV/Mak GP) trimer VLPs (viruslike particles) with a size of 30–40 nm using the Sf9 (engineered *Spodoptera frugiperda*) insect cell-recombinant baculovirus expression system [65]. In mice, adjuvanted with the Matrix-M which consists of two populations of 40 nm ISCOMs: 85% Matrix-A of saponin QH-A fraction +15% Matrix-C of saponin QH-C, EBV/Mak GP VLPs induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8+ T cells as well as effector B cells. Noteworthy, the immunity established in the vaccinated mice conferred a 100% protection against a lethal viral challenge, suggesting the Matrix-M adjuvanted EBV/Mak GP VLP NPs an effective VADS for developing subunit vaccines against the deadly Ebola infections. Similarly, the group using Sf9 insect cell platform engineered a recombinant trivalent NP influenza vaccine (tNIV), which when intramuscularly administered with Matrix-M to ferrets induced high levels of broadly neutralizing antibodies against A (H1N1) strain, B strain and, especially, a panel of all historic (2000–2017) A/H3N2 strains [66]. In particular, in a clinical trial involving 330 adults, the 60-μg dose of tNIV/50 μg Matrix-M induced significantly greater HA inhibition antibody responses against a panel of wild-type A (H3N2) strains than did the inactivated trivalent vaccine Fluzone [67], showing that Matrix-M/tNIV may be an efficient strategy for developing the effective universal influenza vaccines with additional advantage in

tion against HBV infection and as a potential therapeutic vaccine.

avoidance of the mismatching Ags as occurred in conventional procedures.

or malicious neoplasms.

**3. Conclusions**

Summarily, the nanosized cage-like ISCOMs constituted through self-assembly of a combination of saponin, phospholipid and cholesterol are a multifunctional VADS, which can deliver or adjuvant Ags and, in both cases, can enormously boost the efficacy of subunit vaccines. In particular, ISCOMs can be combined with other adjuvants such as TLRas to further improve the immunostimulatory effects for enhancing function of adjuvanted Ags, thus providing a diverse platform for making therapeutic as well as prophylactic vaccines against pathogens

The NP-based VADSs provide an efficient strategy for delivering and enhancing efficacy of subunit vaccines, which are weak immunogens but represent the current trends in the
